曲古菌素A联合细胞因子体外诱导小鼠胚胎干细胞分化为肝细胞

Trichostatin A combined with cytokines induces differentiation of embryonic stem cells into hepatocytes

目的:建立细胞因子和曲古菌素A(trichostatin A,TSA)联合诱导小鼠胚胎干细胞(embryonic stem cell,ESC)向肝细胞分化的3步法.方法:ESC在含激活素A的不含白血病抑制因子的培液中培养3 d分化为定形内胚层细胞;然后加入酸性成纤维细胞生长因子和T S A联合诱导5 d,再加入肝细胞生长因子(hepatocyte growth factor,HGF)等诱导因子培养5 d,向肝细胞方向分化,最后以致瘤素M、地塞米松继续培养5 d形成成熟肝细胞.自发分化组做阴性对照组不加上述因子,不含白血病抑制因子(leukemia inhibitory factor,LIF)的培养液进行自发分化培养.结果:诱导分化18 d后,用光镜、免疫荧光、RT-PCR检测显示诱导18 d后,出现了大量类肝脏细胞表型的上皮细胞,RT-PCR检测显示这些细胞表达成体肝脏细胞特异标记如酪氨酸转氨酶、白蛋白(albumin,ALB)、天冬氨酸转氨酶、甲状腺激素结合蛋白等.细胞免疫荧光也显示为甲胎蛋白、ALB、细胞角蛋白18阳性,分化细胞具有成熟肝脏细胞所具有的糖原储存、吲哚菁绿摄取和释放功能.其分化效率用ALB阳性率表示,诱导分化率可达57.38%.结论:组蛋白去乙酰化酶抑制剂TSA联合细胞因子体外诱导ESC可以成功分化为肝细胞,从而为体外获得大量肝细胞对肝病的临床细胞移植治疗及其研究提供理论和实践基础.

AIM： To present a novel 3-step procedure to efficiently direct the differentiation of mouse embryonic stem cells（ESCs） into hepatocytes. METHODS： Mouse ESCs were first induced to differentiate into definitive endoderm cells by three days of activin A treatment. Next, definitive endoderm cells were induced to efficiently differentiate to hepatocytes in the presence of acid fibroblast growth factor（a FGF） and trichostatin A（TSA） in the culture medium for 5 d. RESULTS： After 10 d of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunofluorescence and RTPCR. Furthermore, these cells were tested for the functions associated with mature hepatocytes including glycogen storage, indocyanine green uptake and release, and the rate of hepatic differentiation was determined by counting the albumin-positive cells, which showed that the rate of hepatic differentiation was 57.38%. CONCLUSION： The method presented in this study provides a new resource for hepatocyte transplantation.